Project Summary Frequent exacerbations induce airway injury and remodeling, and define the severity of asthma. Severe asthma also manifests as a phenotype with irreversible airway obstruction. However, the role of airway remodeling in the pathogenesis of irreversible airway obstruction remains controversial. Transforming growth factor ?1 (TGF-?1) plays a pivotal role in orchestrating airway remodeling; whether TGF-?1 modulates human airway smooth muscle (HASM) shortening and airway hyperresponsiveness (AHR) is unknown. We recently discovered that TGF-?1 alone evokes contraction and augments agonist-induced shortening of HASM, the pivotal cell regulating bronchomotor tone. Our overarching goal is to define the molecular transduction processes that regulate TGF-?1 effects on HASM excitation-contraction (EC) coupling in asthma. We have shown that contractile agonists evoked HASM shortening by activating G12, RhoA, PI3K? and Rho kinase. Whether these processes mediate TGF-?1 effects on EC coupling remain unknown. We posit a central hypothesis that TGF-?1 modulates HASM shortening by activating PI3K?, Rho Kinase and RhoA-dependent actin polymerization in asthma. To test this hypothesis, we developed novel techniques of single cell force generation, human precision cut lung slices (hPCLS), targeted protein knockdown and, human models of AHR. In Aim 1, we will define whether TGF-?1 increases bronchomotor tone and augments agonist-induced HASM shortening in a PI3K?-/Rho kinase-dependent manner. Using asthma- and non-asthma-derived HASM cells and hPCLS, disease-state effects on TGF-?1-induced activation of PI3K? and Rho Kinase will be determined. The role of Smad proteins in mediating TGF-?1-induced single cell contraction and AHR will be characterized after Smad3/4 knockdown. In Aim 2, we will determine whether G12 and RhoA depletion modulates activation of RhoA, PI3K?, ROCK activity and HASM shortening after TGF-?1 exposure. In Aim 3, we will explore whether actin dynamics mediates TGF-?1 effects on HASM contraction. Phosphorylation levels of cofilin and filamentous/globular actin ratios, key modulators of actin polymerization, will be measured in the presence and absence of latrunculin A (an actin disruptor) or siRNA to cofilin. In collaboration with Project 3, Cores A, and B, we will characterize the pivotal signaling pathways mediating the effects of TGF-?1 on bronchomotor tone and AHR, and identify novel therapeutic targets and molecules to prevent or abrogate these effects, which are characteristically observed after asthma exacerbations and/or in severe asthma.